A Rapid imethod Test for Screening Pesticides V.2.0 for Cliquid Software

A Rapid imethod Test for Screening Pesticides V.2.0 for Cliquid Software imethods Test Pesticides Screening Version 2.0 for Cliquid Software With more than 800 pesticides currently in use there is growing concern by government agencies about the effect of pesticide exposure on human health. As a result, the residue level of pesticides in food, water and other consumer products are highly regulated. While analytical techniques like GC or GC/MS have traditionally been used for pesticide analysis, LC/MS/MS has quickly become the technique of choice due to the ability to analyze a more diverse set of pesticides, faster with better sensitivity and less sample preparation. The following description outlines the instrument requirements and expected results obtainable from the AB SCIEX imethod Test for Pesticide Screening and Quantitation using an AB SCIEX 3200 QTRAP system or API 3200 system. This imethod test consists of a pre-configured method to screen for 494 pesticides and individual quantitation methods for carbamate, phenyl urea, triazine, organophosphorus, and acidic pesticides. All methods have also been verified for use on AB SCIEX 4000 QTRAP, API 4000, QTRAP 5500 and AB SCIEX TripleQuad 5500 systems. More in-depth sample preparation, and instrument parameter information is included as part of the standard operating procedure provided with the method, as are the required analytical columns. Solvents, standards and any supplies required for sample preparation are not included. Please note that the use of QTRAP system technology is recommended for use with the screening method provided and that the associated library is not included and must be purchased separately. XIC of +MRM (517 pairs): 207.0/161.0 amu Expected RT: 0.8 I... Max. 75.8 cps. 1.20e5 1.00e5 Intensity, cps 8.00e4 6.00e4 4.00e4 2.00e4 0.00 0.55 0.82 11.03 11.20 11.49 12.12 2 4 6 8 10 12 Time, min Figure 1: Chromatogram of a 303 pesticides mix on an API 3200 LC/MS/MS system (10 ng/ml) p 1

The pesticide screening method included in this imethod test is for the routine screening of 494 pesticides from food samples using the QuEChERS extraction and cleanup technique. The method uses external calibration standards and matrix spike recoveries to correct for sample and instrument variability and is based upon the use of a 3200 QTRAP LC/MS/MS system. A suggested method for the extraction of pesticides from vegetable, nut and citrus plant materials is also provided and is based upon the use of Restek QuEChERS tubes. Separation is achieved using a simple methanol / water gradient with ammonium formate with a Phenomenex Synergi 2.5 µm Fusion 100 Å, 50 x 2 mm HPLC column that is included with this imethod test. Table 1 provides representative recoveries, signal-to-noise ratios and estimated detection limits for 130 selected pesticides spiked at 25 ng/ml in a cucumber matrix using QuEChERS sample processing technique. Table 1. Representative recoveries, signal-to-noise ratios and estimated detection limits for 130 selected pesticides spiked at 25 ng/ml in a cucumber matrix using QuEChERS sample processing, as run on the 3200 QTRAP LC/MS/MS system Analyte % Recovery S/N * % CV Estimated detection limit (ng/ml) Aldicarb-sulfoxide 97.0% 26 4.2% 9.6 Aldoxycarb 96.4% 33.2 10.0% 7.5 Alloxydim 60.8% 32.2 7.5% 7.8 Aminocarb 84.2% 84 2.6% 3.0 Atrazine 65.1% 34 2.4% 7.4 Atrazine-2-hydroxy 79.9% 77.8 2.9% 3.2 Atrazine-desethyl-2-hydroxy 104.1% 38.8 1.9% 6.4 Benalaxyl 94.9% 39.5 18.1% 6.3 Bendiocarb 120.2% 33.1 16.2% 7.6 Benzoximate 119.9% 29.3 10.0% 8.5 Boscalid 15.3% 14.3 18.7% 17.5 Bromacil 99.0% 15.9 8.4% 15.7 Bupirimate 106.2% 43.8 9.0% 5.7 Butocarboxim-sulfoxid 102.2% 37.7 2.3% 6.6 Buturon 98.6% 20.4 4.1% 12.3 Carbaryl 98.4% 44.7 1.5% 5.6 Carbetamide 94.3% 58.8 4.9% 4.3 Chloridazon 111.1% 35.5 1.7% 7.0 Chlorotoluron 149.2% 38.1 2.4% 6.6 Chloroxuron 69.5% 24.7 11.0% 10.1 Cinosulfuron 88.6% 28.8 18.9% 8.7 Clomazone 107.2% 42.3 1.8% 5.9 Cloquintocet-mexyl 140.8% 95.8 10.1% 2.6 Coumaphos 120.8% 24.4 3.6% 10.2 Cyanazine 115.6% 22.1 20.1% 11.3 Cyproconazole 48.6% 24.6 7.1% 10.2 Cyprodinil 120.2% 11.3 7.6% 22.1 Demeton-S-methyl-sulfone 90.0% 32.8 1.9% 7.6 Diazinon 100.5% 81.9 7.2% 3.1 Diclobutrazol 88.3% 26.3 7.6% 9.5 Dicrotophos 112.7% 58.1 19.1% 4.3 Diethofencarb 28.5% 16.2 8.0% 15.4 Difenoxuron 104.0% 51 5.3% 5.0 Dimefuron 92.5% 20 6.6% 12.6 Dimethachlor 88.8% 13 11.8% 19.5 Dimethenamide 100.5% 39 2.7% 6.5 Dimetilan 106.0% 101 2.5% 2.5 Dodemorph 67.0% 107 3.0% 2.3 Ethidimuron 139.0% 28 6.5% 8.9 Ethiofencarb-sulfon 113.0% 59 14.7% 4.3 Ethiofencarb-sulfoxid 99.1% 119 3.6% 2.1 p 2

Analyte % Recovery S/N * % CV Estimated detection limit (ng/ml) Ethion 113.9% 17 7.2% 14.8 Ethirimol 99.6% 42 4.9% 6.0 Ethoprophos 112.9% 16 14.1% 15.2 Etrimfos 93.3% 64 4.6% 3.9 Fenbuconazole 93.0% 21 2.2% 11.9 Fenfuram 98.0% 46 7.7% 5.5 Fenhexamid 47.9% 15 15.1% 17.0 Fenoxaprop-P-ethyl 113.9% 32 7.2% 7.8 Fenpropimorph 130.8% 152 4.2% 1.6 Fenpyroximate 161.6% 15 8.4% 16.8 Fenuron 102.8% 47 5.5% 5.3 Flamprop-M-isopropyl 104.1% 55 4.1% 4.5 Flamprop-M-methyl 90.2% 20 3.7% 12.4 Flazasulfuron 100.8% 74 7.5% 3.4 Fluazifop-butyl 144.5% 50 3.6% 5.0 Flufenacet 89.7% 22 12.8% 11.5 Fluometuron 120.9% 77 2.3% 3.2 Fluridone 68.4% 30 11.8% 8.4 Flurtamone 89.7% 20 5.2% 12.3 Fuberidazole 102.7% 125 6.2% 2.0 Haloxyfop-P-methyl 106.4% 15 3.2% 16.9 Hexaconazole 100.9% 23 2.1% 11.0 Hexazinone 106.3% 202 15.8% 1.2 Imazalil 117.7% 34 18.1% 7.3 Isazofos 74.9% 13 8.0% 19.1 Isoprothiolane 62.1% 77 1.2% 3.2 Lenacil 111.3% 43 6.1% 5.8 Malaoxon 44.7% 50 9.5% 5.0 Malathion 94.7% 30 5.9% 8.3 Mepanipyrim 79.5% 3 7.9% 80.6 Metconazole 118.2% 19 7.3% 13.4 Methamidophos 90.9% 33 6.5% 7.6 Methfuroxam 66.2% 96 1.1% 2.6 Methidathion 47.9% 35 4.2% 7.2 Metobromuron 116.1% 19 4.0% 12.9 Metolachlor 82.7% 22 2.6% 11.4 Metosulam 99.7% 51 2.7% 4.9 Monuron 100.3% 22 2.8% 11.6 Napropamide 103.7% 31 11.5% 8.1 Oxasulfuron 87.8% 78 6.6% 3.2 Oxycarboxin 98.5% 110 5.3% 2.3 Paclobutrazol 105.4% 48 9.2% 5.2 Phosalone 102.9% 13 9.0% 18.9 Phosphamidon 95.2% 104 6.5% 2.4 Picoxystrobin 133.1% 104 3.8% 2.4 Pirimiphos-ethyl 129.7% 57 4.1% 4.4 Pirimiphos-methyl 105.1% 63 3.1% 4.0 Prometon 100.5% 105 6.0% 2.4 Prometryne 49.5% 14 19.5% 18.4 Propamocarb 106.5% 20 1.9% 12.8 Propazin-2-hydroxy 81.5% 19 3.6% 12.9 Propazine 72.8% 11 10.7% 22.5 Prosulfocarb 114.9% 145 6.1% 1.7 Pymetrozine 101.4% 115 2.6% 2.2 p 3

Analyte % Recovery S/N * % CV Estimated detection limit (ng/ml) Pyraclostrobin 141.1% 24 9.6% 10.5 Pyrazophos 108.9% 55 4.8% 4.5 Pyridaben 102.8% 18 4.1% 14.1 Pyridaphenthion 88.6% 10 6.3% 25.3 Pyrimethanil 40.1% 18 5.8% 13.6 Pyriproxyfen 153.8% 51 4.3% 4.9 Quinalphos 124.9% 17 7.3% 14.8 Quinmerac 104.9% 116 1.3% 2.2 Sebuthylazine 56.9% 55 3.6% 4.6 Sebuthylazine-desethyl 88.6% 39 7.1% 6.5 Sethoxydim 82.5% 28 10.1% 8.9 Siduron 59.7% 14 5.3% 17.5 Simazine-2-hydroxy 105.5% 99 10.6% 2.5 Simetryn 95.7% 96 6.6% 2.6 Spiroxamine 97.1% 230 6.0% 1.1 Sulfometuron-methyl 98.2% 103 5.1% 2.4 Tebuconazol 87.3% 45 9.6% 5.5 Tebufenpyrad 125.2% 13 10.6% 18.8 Tebutam 87.2% 22 7.3% 11.4 Tebuthiuron 97.8% 61 14.4% 4.1 Terbufos 44.3% 4 20.3% 65.8 Terbuthylazine 31.7% 18 3.3% 14.1 Terbuthylazine-desethyl 88.7% 39 8.1% 6.5 Terbutryn 31.9% 119 6.0% 2.1 Tetrachlorvinphos 101.3% 20 8.7% 12.5 Tetraconazole 82.1% 32 16.3% 7.8 Thifensulfuron-methyl 100.8% 74 4.2% 3.4 Triasulfuron 80.5% 35 7.5% 7.2 Triazophos 31.9% 19 9.9% 13.2 Tricyclazole 93.8% 72 1.9% 3.5 Trietazine 75.4% 12 13.6% 21.0 Trifloxystrobin 113.9% 83 5.8% 3.0 Triflumizole 122.4% 25 11.9% 10.1 Triticonazole 117.1% 24 5.8% 10.6 Uniconazole 66.5% 24 24.0% 10.6 Vamidothion 113.1% 36 13.9% 7.0 * Signal-to-noise obtained at three standard deviations on a 3200 QTRAP System. Estimated detection limit based on estimated signal at 10 x the Signal-to-noise ratio. Quantitation Methods The following information outlines the list of standard compounds that in each of the quantitation methods for each compound class listed above. The methodology presented below was developed to quantify each pesticide when run by compound class, at < 10 ppb in order to meet regulatory requirements. The 603-compound MRM catalogue provided can also be used to create new customized tests according to any combinations required. Example sample preparation procedures are provided for fruits and vegetables, based upon a simple sample homogenization, centrifugation, extraction and dilution. These procedures may require additional optimization based upon the actual composition and consistency of the fruit or vegetable under investigation. Deuterated and/or C13-labeled internal standards of known concentrations are added during sample preparation to monitor sample recovery. p 4

Figure 2: Chromatogram of a 300 pesticides mix spiked by API 3200 LC/MS/MS system (100 ng/ml) XIC of +MRM (34 pairs): 253.2/171.2 Da from Sample 1 (5 ng/ml) of Triazines.wiff (Turbo Spray) Max. 7.5e4 cps. 1.3e5 1.3e5 1.3e5 1.2e5 1.2e5 1.1e5 1.1e5 1.0e5 9.5e4 9.0e4 8.5e4 8.0e4 Intensity, cps 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 5000.0 0.0 Triazines 6.9 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Time, min Figure 3: Chromatogram of triazine pesticides at 5 ng / ml p 5

Table 2: List of triazine pesticides with retention times for each Triazines RT ( Min) Atrazine 8.5 Ametryn 7.8 Cyanazine 6.7 Desisopropyl-atrazine 5.4 Desethyl-atrazine 4.0 Hexazinon 6.9 Metazoachlor 7.8 Metolachlor 9.2 Metribuzin 7.4 Prometon 8.3 Prometryn 9.0 Propazine 8.6 Sebutylazine 8.4 Simazine 6.9 Terbuthylazine 8.6 Terbutryn 9.1 D5-atrazine (internal std) 7.7 XIC of +MRM (32 pairs): 229.2/172.4 Da from Sample 1 (20 ng/ml) of Phenyl ureas.wiff (Turbo Spray) Max. 3.0e5 cps. 9.5e5 9.0e5 Phenylurea Pesticides 8.5e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 Intensity, cps 5.5e5 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 7.0 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 0.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 Time, min Figure 4: Chromatogram of phenyl urea pesticides at 20 ng/ml p 6

Table 3: List of phenyl urea pesticides with retention times for each Phenyl Urea RT ( Min) Chlortoluron 7.6 Diflubenzuron 9.3 Diuron 8.0 Fenuron 4.6 Fluomethuron 7.6 Isoproturon 7.9 Linuron 8.5 Metabenzthiazuron 7.6 Metobromuron 7.7 Metoxuron 6.2 Monolinuron 7.4 Propanil 8.5 Siduron 8.6 Tebuthiuron 7.1 Thidiazuron 7.0 D6-Isoproturon (internal std) 7.6 XIC of +MRM (94 pairs): 239.2/72.1 Da from Sample 1 (10ng/mL) of Carbamates.wiff (Turbo Spray) 3.4e5 3.2e5 Max. 2.2e5 cps. Carbamates 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 7.4 Intensity, cps 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 Time, min Figure 5: Chromatogram of carbamate pesticides at 10 ng/ml p 7

Table 4: List of carbamate pesticides with retention times for each Carbamates RT ( Min) 3-Hydroxycarbofuran 5.0 Alanycarb 9.4 Aldicarb 6.0 Aldicarb-sulfone 2.2 Aldicarb-sulfoxide 1.6 Aminocarb 6.7 Bendiocarb 6.9 Benfuracarb 10.1 Benomyl 9.6 Butocarboxim 5.9 Butocarboxim-sulfoxide 1.5 Butoxycarboxim 2.1 Carbaryl 7.2 Carbofuran 6.9 Carbosulfan 11.5 Chloropropham 8.8 Dimepiperate 9.8 Dioxacarb 4.8 EPTC (S-Ethyl N,Ndipropylthiocarbamate) 9.2 Esprocarb 10.1 Ethiofencarb 7.4 Ethiofencarb-sulfone 4.2 Ethiofencarb-sulfoxide 4.2 Fenobucarb 8.3 Fenothiocarb 9.2 Fenoxycarb 9.2 Furathiocarb 10.1 Iprovalicarb 9.0 Isoprocarb 7.6 Methiocarb 8.4 Methocarb-sulfone 5.5 Methomyl 2.9 Oxamyl 2.3 Phenmedipham 8.2 Primicarb 7.5 Promecarb 8.5 Propamocarb 3.2 Propoxur 6.8 Pyributicarb 10.3 Terbucarb 9.8 Thiobencarb 9.6 Thiodicarb 7.5 Thiofanox 7.5 Thiofanox-sulfone 4.7 Thiram 6.7 Triallate 10.4 XMC 7.3 p 8

XIC of +MRM (68 pairs): 305.0/169.0 Da from Sample 1 (10ppb) of Organophosphorus.wiff (Turbo Spray) Max. 9.1e5 cps. 9.0e5 8.5e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 Intensity, cps 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 Organophosphorus Pesticides 13.2 0.0 2 4 6 8 10 12 14 16 Time, min Figure 6: Chromatogram of organophosphorus pesticides at 10 ng/ml Table 5: List of organophosphorus pesticide with retention times for each Organophosphorus RT ( Min) Acephate 3.1 Azinphos ethyl 12.6 Azinphos methyl 11.6 Chlorfenvinphos 13.1 Chlorpyrifos 14.6 Coumaphos 13.3 Demeton-s-methyl 8.5 Demeton-S-methyl-sulfone 7.3 Diazinon 13.2 Dichlorvos 9.9 Dicrotophos 7.8 Dimethoate 8.5 Famphur 11.1 Fenthion 13.2 Malathion 12.3 Mevinphos 9.1 Naled (Dibrom) 11.3 Parathion-ethyl (Parathion) 13.1 Parathion-methyl 7.3 Phorate 13.4 Phorate sulfoxide 10.8 Pirimiphos-ethyl 14.2 Pirimiphos-methyl 13.5 Sulfotep 13.1 Sulprofos 14.4 p 9

Organophosphorus RT ( Min) Tetrachlorvinphos 12.8 Triazophos 12.4 Tokuthion (Prothiophos) 15.1 EPTC (Internal std) 12.9 XIC of -MRM (38 pairs): 239.0/193.0 Da from Sample 1 (10ppb) of Acidic pesticides.wiff (Turbo Spray) Max. 2.0e5 cps. 2.6e5 2.5e5 2.4e5 2.3e5 2.2e5 2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 Intensity, cps 1.5e5 1.4e5 1.3e5 1.2e5 Acidic Pesticides 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Figure 7: Chromatogram of acidic pesticides at 10 ng/ml Table 6: List of acidic pesticides with MRM transitions and retention times for each Acidic Pesticides RT ( Min) 2,4,5-T 10.5 2,4,5-TP 11.5 2,4-D 8.6 2,4-DB 11.8 2,4-DP (Dicloprop) 10.2 Bentazon 6.5 Bromoxynil 8.8 Clopyralid 3.4 Dicamba 5.6 Dinoterb 12.1 Dinoseb 12.1 Fluoroxypyr 6.6 Imazapyr 4.7 Ioxynil 10.2 MCPA 9.0 MCPB 11.9 MCPP 10.6 PCP 12.8 Triclopyr 9.6 12.1 p 10

Please note that the results presented above were obtained using a single instrument and single set of standards and samples. Prior to production use, the method should be fully validated with real samples, and the results here may not be typical for all instruments. Variations in LC column properties, chemicals, environment, instrument performance and sample preparation procedures will impact performance, thus these results should be considered as informative rather than representative. System Requirements In order to run this method as outlined above, the following equipment and reagents are required: An AB SCIEX 3200 Series (3200 QTRAP or API 3200 ), 4000 Series (4000QTRAP or API 4000 ), AB SCIEX QTRAP 5500 or AB SCIEX Triple Quad 5500 LC/MS/MS system A Shimadzu Prominence 20A LC System with reservoir tray and bottles, system controller CBM-20A, 100 µl mixer, 2 isocratic pumps LC-20AD, 3 channel degasser autosampler SIL-20AC, column oven CTO-20AC or Agilent 1100/1200 LC system with binary pump G1312A (without static mixer), well plate autosampler, and thermostated column oven. Pesticide Standards (www.sigmaaldrich.com) Pesticide Internal Standards (www.sigmaaldrich.com) LC/MS-grade water, methanol, acetonitrile, ammonium acetate, ammonium formate Phenomenex Synergi 4µ Fusion-RP 80A 50 x 2 mm HPLC column for carbamates and triazines Phenomenex Synergi 2.5 µm Fusion 100 Å, 50 x 2 mm HPLC column for the pesticide screening method 1.5 ml Eppendorf tubes A Centrifuge, able to accommodate Eppendorf tubes and run at 14000 rpm Pipettes and standard laboratory glassware Both Phenomenex Fusion HPLC columns come standard with this imethod test kit. This method can also be run on other HPLC systems, given that they are supported for use by the Cliquid Software and the retention times are updated to reflect the configuration used. Ordering Information Product Name imethods Test for Pesticide Screening V.2.0 for Cliquid Software Part Number 1037144 While the information provided above outlines the instrument requirements and expected results obtainable from the AB SCIEX imethod Test for Pesticide Screening, the results obtained do require some experience with LC/MS/MS and sample preparation procedures. As such, web-based and on-site training are available to assist in the deployment of the imethod test and are recommended for inexperienced users. Please consult your local sales representative for more details. Important Note The purchase and use of certain chemicals listed above may require the end user to possess any necessary licenses, permits or approvals, if such are required in accordance with local laws and regulations. It is the responsibility of the end user to purchase these chemicals from a licensed supplier, if required in accordance with local laws and regulations. The suppliers and part numbers listed below are for illustrative purposes only and may or may not meet the aforementioned local requirements. Legal Acknowledgements/Disclaimers The imethod Test described above has been developed by AB SCIEX to provide all the sample prep and instrument parameters required to accelerate the adoption of this method for routine testing. The performance of this method will need to be verified in a given lab due to potential variations in instrument performance, maintenance, chemicals and procedures used, technical experience, sample matrices and environmental conditions It is the responsibility of the end user to make adjustments to this method to account for slight differences in equipment and/or materials from lab to lab as well as to determine and validate the performance of this method for a given instrument and sample type. Aworking knowledge of Analyst Software may be required to do so. p 11

The suppliers identified in this document are provided for information purposes only and is not intended to be an exhaustive representation of all manufacturers or suppliers of the referenced product. AB SCIEX makes no warranties or representations as to the fitness or the continued fitness of a specific product by any of the manufacturers/suppliers referenced herein or the supplier. AB SCIEX assumes no responsibility or contingent liability, including indirect or consequential damages, for any use to which the purchaser may put the referenced suppliers products, or for any adverse circumstances arising therefrom. For Research Use Only. Not for use in diagnostic procedures. 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Publication number: 1160210-02 p 12